Abstract Chronic kidney disease affects over 26 million Americans. For the one million patients with end stage renal disease, dialysis and kidney transplant are the only therapeutic options. However, dialysis is palliative and kidney donors are in short supply. Thus, there is a critical need for new therapeutic strategies. The basic unit of the kidney is the nephron, a highly vascularized filtration and recovery unit. In the nephron, the plasma filtrate generated in the glomerulus passes into the proximal tubule (PT). The PT is lined by cuboidal proximal tubule epithelial cells (PTECs), the major resorptive cell type of the nephron characterized by the polarized distribution of channels and transporters that recover essential molecules and ions from the plasma filtrate. In acute kidney injury, PTECs are highly susceptible to damage. Surviving PTECs can repair the injured nephron, but endogenous repair mechanisms are not well-understood. This slows the development of new therapeutic strategies to accelerate PT repair in both acute and chronic kidney disease. Recently, the McMahon lab identified that the transcription factor SOX9 is up-regulated in PTECs after acute kidney injury in mice. This SOX9+ population of PTECs repopulates the nephron and restores function. However, whether a similar mechanism underlies repair of the human PT is unclear. One of the only practical approaches to identify mechanisms of human PT regeneration is to study human PTECs cultured in vitro. However, conventional culture substrates are highly artificial and lack physical cues present in the native PT that impact PTEC phenotype and survival, such as fluid shear stress. As a result, PTECs in conventional 2-D culture lose polarity and functionality. Recently, ?Organ on Chip? approaches have been developed to expose PTECs in vitro to physical cues similar to those in vivo, such as fluid shear stress. PTECs cultured within these platforms form differentiated structures and have improved functionality. However, existing platforms require specialized equipment that is not accessible to most research groups, neglect to include supporting cell populations (such as endothelial cells), and have not been used as tools for identifying mechanisms of PT regeneration. Thus, in Aim 1, we will use off-the-shelf equipment to engineer a scalable platform for engineering and maintaining a human PT, leveraging the McCain lab?s experience in engineering ?Organ on Chip? models of striated muscle. Our key design parameters are to apply fluid shear stress to primary human PTECs cultured as a tubule within a protein-derived extracellular matrix (ECM) hydrogel with relatively low elastic modulus. After validating that our engineered PT recapitulates key structural and functional phenotypes, we will add supporting cell populations (endothelial cells, fibroblasts) into the ECM hydrogel and establish any further improvements in PTEC viability, structure, and/or function. In Aim 2, we will induce global and local injury to our engineered PT and examine the expression of SOX9 throughout the PT during repair. We will then determine whether manipulating SOX9 activity can augment PT repair. This project is especially well-suited for the EBRG funding mechanism because we have established a multidisciplinary team (Prof. Megan McCain: junior investigator in biomedical engineering; Prof. McCain Andy McMahon: established investigator in kidney development) to develop a new engineered PT tissue platform to enable our hypothesis-driven research into SOX9-mediated mechanisms of human PT regeneration.